OBJECTIVE This study was to investigate the effects of CP-25 on the functions of activated human B cells through regulating BAFF and TNF-alpha signaling.METHODS B cells from peripheral blood mononuclear cells(PBMCs) of normal human were isolated using magnetic cell separation(MACS) by a positive selection.B cells(107 cells·mL~(-1)) were stimulated by BAFF(100 ng·mL~(-1))or TNF-alpha(100 ng·mL~(-1)) for two hours,and then were treated with CP-25(10-5 mol·L~(-1)) or Rituximab(5 μg·mL~(-1)) or Etanercept(10 μg·mL~(-1)).B cell proliferation was detected by CCK-8.B cell subsets and BAFF receptors(BAFFR,BCMA and TACI) were analyzed by flow cytometry.The expression of TNFR1 and TNFR2 on B cells was analyzed by flow cytometry.The expression of MKK3,MKK6,P-p38,P-p65,TRAF2 and p100/52 was analyzed by Western blotting.RESULTS CP-25 inhibited B cells proliferation stimulated by BAFF or TNF-alpha.CP-25,Rituximab and Etanercept reduced the percentage and numbers of CD19~+B cells,CD19~+CD20~+B cells,CD19~+CD27~+B cells and CD19~+CD20~+CD27~+B cells induced by BAFF or TNF-alpha.CP-25 down-regulated the high expression of BAFFR,BCMA and TACI stimulated by BAFF or TNF-alpha.CP-25,Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha.CP-25,Rituximab and Etanercept down-regulated the expression of MKK3,P-p38,P-p65,TRAF2 and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha.CONCLUSION CP-25 regulated moderately activated B cells function by by regulating the classical and alternative NF-κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway.This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug. OBJECTIVE This study was to investigate the effects of CP-25 on the functions of activated human B cells through regulating BAFF and TNF-alpha signaling. METHODS B cells from peripheral blood mononuclear cells (PBMCs) of normal human were isolated using magnetic cell separation MACS) by a positive selection.B cells (107 cells · mL -1) were stimulated by BAFF (100 ng · mL -1) or TNF-alpha (100 ng · mL -1) for two hours, and then were treated with CP-25 (10-5 mol·L -1) or Rituximab (5 μg · mL -1) or Etanercept (10 μg · mL -1) ). B cell proliferation was detected by CCK-8.B cell subsets and BAFF receptors (BAFFR, BCMA and TACI) were analyzed by flow cytometry. The expression of TNFR1 and TNFR2 on B cells was analyzed by flow cytometry. The expression of MKK3 , MKK6, P-p38, P-p65, TRAF2 and p100 / 52 were analyzed by Western blotting.RESULTS CP-25 inhibited B cells proliferation stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept reduced the percentage and numbers of CD19 ~ + B cells, CD19 ~ + CD20 ~ + B cells, CD19 + CD27 + B cells and CD19 + CD20 + CD27 + B cells induced by BAFF or TNF-alpha. CP-25 down-regulated the high expression of BAFFR, BCMA and TACI stimulated by BAFF or TNF-alpha.CP-25, Rituximab and Etanercept down-regulated significantly the expression of TNFR1 and TNFR2 on B cell stimulated by BAFF or TNF-alpha. CP-25, Rituximab and Etanercept down-regulated the expression of MKK3, P-p38 , P-p65, TRAF2 and p52 in B cells stimulated by BAFF and the expression of TRAF2 and P-p65 in B cells stimulated by TNF-alpha. CONCLUSION CP-25-regulated by genetically activated B cells function by by the classical and alternative NF -κB signaling pathway mediated by BAFF and TNF-alpha-TRAF2-NF-κB signaling pathway. This study suggests that CP-25 may be a promising anti-inflammatory immune and soft regulation drug.